Pulmonary arterial hypertension (PAH) results from vasoconstriction and vascular remodeling. Our goal is to understand the role lung side population (SP) progenitor cells play in the development of hypobaric hypoxia- induced PAH. We have developed an isolation/enrichment procedure that is based on the ability of primitive progenitor cells to efflux a Hoechst dye via an ABCG2 transporter which is common to adult stem cells, termed the side population (SP). We localized the lung SP as associated with the alveolar-capillary network in mouse lung. (Specific Aim 1) Their stem cell nature was demonstrated by our ability to repeatedly isolate them from lung tissue, maintain them in cell culture for a prolonged period of time and induce them to differentiate into endothelial (EC) or myofibroblast cell lineages. Differentiation was demonstrated by studies that show the induction of VE-cadherin and angiogenic potential. Myofibroblasts were characterized by collagen/elastin synthesis. (Specific Aim 2) Additional studies in our lab demonstrated that augmentation of the lung SP in mice, combined with hypobaric hypoxia exposure, increase ventricular systolic pressure. Following these hemodynamic studies the lungs were analyzed to localize the lung SP cells. These exogenously administered lung SP cells were found in the lung associated lymph nodes and parenchyma. (Specific Aim 3) Platelet- derived growth factor B (PDGF-BB) is required for our in vitro myofibrobalst differentiation of lung SP and is elevated in the lungs of our mice with hypobaric hypoxia induced PAH. In the presence of PDGF-BB/abl kinase inhibitor (Gleevec), we could inhibit myofibroblast differentiation in vitro and decrease elevated right ventricular systolic pressure associated with augmented lung SP in vivo. We will use a lineage tracing strategy to examine the potential of endogenous lung SP cells during PAH in vivo as a function of the transcription factor, FoxO1. We will examine the hypoxia-dependent vasoconstrictive potential of lung SP cells by quantifying muscularization of small caliber vessels and SP production of endothelin-1 in vivo and in vitro and using isolated perfused lung analyses. Additional studies will determine whether inhibition of PDGFR2/abl kinase signaling decreases hypoxia-induced SP cell proliferation and differentiation or the vasoconstrictive effects of lung SP on the progression of PAH thru downstream PI3K signaling and regulation of the FoxO1 activity. PUBLIC HEALTH RELEVANCE: Pulmonary hypertension, chronic obstructive pulmonary disease, interstitial pulmonary fibrosis and other adult lung conditions are a major cause of morbidity and mortality. Deaths due to these conditions have doubled in the last decade. There is an increasing emphasis on the development of cell-based therapies to address these conditions, but the lung is a recalcitrant candidate for these strategies because of the diverse cell types and functions as well as a lack of understanding of how chronic disease processes affect stem cell differentiation. Therefore, prior to testing cell-based therapy, it is desirable to use a pre-clinical animal model of PAH to determine how changes in the lung tissue during the development of disease affect resident stem cell differentiation and function.